1. Recombinant DNA Techniques chapter 19
Part I

2. techniques and their applications.
Restriction Digest (to be done in lab)
Southern Blot
Northern Blot
Western Blot
Cloning

3. 1. Restriction Digest: The Cutting of DNA
Endonucleases =
Restricition Enzymes

6. Ladder, marker or standard of known DNA sizes
EXAMPLES OF DNA AGAROSE GELS
Ladder, marker or standard of known DNA sizes

7. An Application of Restriction Digest Chromosomal Disorders

8. Put your thinking cap on!

9. 2 3 Wild type Hb Sickle cell Hb Restriction enzyme Restriction enzyme
1 band is too small to view here) 2 3
Restriction enzyme
loss of cutting site

10. Wt= wildtype
M= mutated
M M
Wt Wt
Wt M

11. An Application to Forensic & Paternity Studies
2. The Southern Blot

12. 1. Restriction Digest & gel electrophoresis
TRANSFER
DNA crime scene
DNA suspect 2
DNA suspect 1
DNA victim

13. You must understand the
reason for the “probe”!

14. But there are thousands of fragments!

15. So sometimes you have to really search for the differences between DNA.

16. Probe is complementary “hot” DNA

17. From: Restriction digest
To result
SUMMARY OF A SOUTHERN BLOT

18. Victim
Suspect 1
Suspect 2
Crime scene
Back to our crime scene

19. Techniques also are available to measure RNA and Protein
3. Northern Blot for RNA
4. Western Blot for Protein

20. Summary: Blotting techniques with probes
Western
Southern
Northern

21. 5. Technique: Cloning of specific genes or DNA fragments
= restriction enzyme +
= ligase

22. Cut vector
Cut foreign DNA or gene
Paste together

23. 2 different antibiotic resistant genes
Bacteria are now resistant to 2 antibiotics

24. Let’s apply these concepts to plasmid transformation.

25. Cut DNA here &
insert foreign DNA

26. Will your transformed colonies with the insert be white or blue?

27. Summary of cloning DNA into a blue/white selection vector

28. End of part 1